Visceral leishmaniasis (VL) is a fatal disease for humans, and no vaccine is currently available. Sand fly salivary proteins have been associated with protection against cutaneous leishmaniasis. To test whether vector salivary proteins can protect against VL, a hamster model was developed involving intradermal inoculation in the ears of 100,000 Leishmania infantum chagasi parasites together with Lutzomyia longipalpis saliva to mimic natural transmission by sand flies. Hamsters developed classical signs of VL rapidly, culminating in a fatal outcome 5-6 months postinfection. Saliva had no effect on the course of infection in this model. Immunization with 16 DNA plasmids coding for salivary proteins of Lu. longipalpis resulted in the identification of LJM19, a novel 11-kDa protein, that protected hamsters against the fatal outcome of VL. LJM19-immunized hamsters maintained a low parasite load that correlated with an overall high IFN-␥/TGF-␤ ratio and inducible NOS expression in the spleen and liver up to 5 months postinfection. Importantly, a delayed-type hypersensitivity response with high expression of IFN-␥ was also noted in the skin of LJM19-immunized hamsters 48 h after exposure to uninfected sand fly bites. Induction of IFN-␥ at the site of bite could partly explain the protection observed in the viscera of LJM19-immunized hamsters through direct parasite killing and/or priming of anti-Leishmania immunity. We have shown that immunity to a defined salivary protein (LJM19) confers powerful protection against the fatal outcome of a parasitic disease, which reinforces the concept of using components of arthropod saliva in vaccine strategies against vector-borne diseases.antisaliva immunity ͉ Leishmania ͉ sand fly saliva ͉ vector-based vaccine

Endogenous regulatory T (Treg) cells are involved in the control of infections, including Leishmania infection in mice. Leishmania viannia braziliensis is the main etiologic agent of cutaneous leishmaniasis (CL) in Brazil, and it is also responsible for the more severe mucocutaneous form. Here, we investigated the possible involvement of Treg cells in the control of the immune response in human skin lesions caused by L. viannia braziliensis infection. We show that functional Treg cells can be found in skin lesions of patients with CL. These cells express phenotypic markers of Treg cells--such as CD25, cytotoxic T lymphocyte-associated antigen 4, Foxp3, and glucocorticoid-induced tumor necrosis factor receptor--and are able to produce large amounts of interleukin-10 and transforming growth factor- beta . Furthermore, CD4+CD25+ T cells derived from the skin lesions of 4 of 6 patients with CL significantly suppressed in vitro the phytohemagglutinin-induced proliferative T cell responses of allogeneic peripheral-blood mononuclear cells (PBMCs) from healthy control subjects at a ratio of 1 Treg cell to 10 allogeneic PBMCs. These findings suggest that functional Treg cells accumulate at sites of Leishmania infection in humans and possibly contribute to the local control of effector T cell functions.

BackgroundSand flies deliver Leishmania parasites to a host alongside salivary molecules that affect infection outcomes. Though some proteins are immunogenic and have potential as markers of vector exposure, their identity and vector specificity remain elusive.Methodology/Principal FindingsWe screened human, dog, and fox sera from endemic areas of visceral leishmaniasis to identify potential markers of specific exposure to saliva of Lutzomyia longipalpis. Human and dog sera were further tested against additional sand fly species. Recombinant proteins of nine transcripts encoding secreted salivary molecules of Lu. longipalpis were produced, purified, and tested for antigenicity and specificity. Use of recombinant proteins corresponding to immunogenic molecules in Lu. longipalpis saliva identified LJM17 and LJM11 as potential markers of exposure. LJM17 was recognized by human, dog, and fox sera; LJM11 by humans and dogs. Notably, LJM17 and LJM11 were specifically recognized by humans exposed to Lu. longipalpis but not by individuals exposed to Lu. intermedia.Conclusions/SignificanceSalivary recombinant proteins are of value as markers of vector exposure. In humans, LJM17 and LJM11 emerged as potential markers of specific exposure to Lu. longipalpis, the vector of Leishmania infantum chagasi in Latin America. In dogs, LJM17, LJM11, LJL13, LJL23, and LJL143 emerged as potential markers of sand fly exposure. Testing these recombinant proteins in large scale studies will validate their usefulness as specific markers of Lu. longipalpis exposure in humans and of sand fly exposure in dogs.

BackgroundSand fly saliva has an array of pharmacological and immunomodulatory components, and immunity to saliva protects against Leishmania infection. In the present study, we have studied the immune response against Lutzomyia intermedia saliva, the main vector of Leishmania braziliensis in Brazil, and the effects of saliva pre-exposure on L. braziliensis infection employing an intradermal experimental model.Methodology/principal findingsBALB/c mice immunized with L. intermedia salivary gland sonicate (SGS) developed a saliva-specific antibody response and a cellular immune response with presence of both IFN-γ and IL-4. The inflammatory infiltrate observed in SGS-immunized mice was comprised of numerous polymorphonuclear and few mononuclear cells. Mice challenged with live L. braziliensis in the presence of saliva were not protected although lesion development was delayed. The inoculation site and draining lymph node showed continuous parasite replication and low IFN-γ to IL-4 ratio, indicating that pre-exposure to L. intermedia saliva leads to modulation of the immune response. Furthermore, in an endemic area of cutaneous leishmaniasis, patients with active lesions displayed higher levels of anti-L. intermedia saliva antibodies when compared to individuals with a positive skin test result for Leishmania.ConclusionThese results show that pre-exposure to sand fly saliva plays an important role in the outcome of cutaneous leishmaniasis, in both mice and humans. They emphasize possible hurdles in the development of vaccines based on sand fly saliva and the need to identify and select the individual salivary candidates instead of using whole salivary mixture that may favor a non-protective response.

Background: Leishmaniasis remains a serious public health problem in several parts of the developing world. Effective prophylactic measurements are hampered by imprecise comprehension of different aspects of the disease, including its immunoregulation. A better comprehension of immunoregulation in human VL may be useful both for designing and evaluating immunoprophylaxis.

Leishmania spp. cause a broad spectrum of diseases collectively known as leishmaniasis. Leishmania braziliensis is the main etiological agent of American cutaneous leishmaniasis (ACL) and mucocutaneous leishmaniasis. In the present study, we have developed an experimental model of infection that closely resembles ACL caused by L. braziliensis. In order to do so, BALB/c mice were infected in the ear dermis with 10 5 parasites and distinct aspects of the infection were evaluated. Following inoculation, parasite expansion in the ear dermis was accompanied by the development of an ulcerated dermal lesion which healed spontaneously, as seen by the presence of a scar. Histological analysis of infected ears showed the presence of a mixed inflammatory infiltrate consisting of both mononuclear and polymorphonuclear cells. In draining lymph nodes, parasite replication was detected throughout the infection. In vitro restimulation of draining lymph node cells followed by intracellular staining showed an up-regulation in the production of gamma interferon (IFN-␥) and in the frequency of IFN-␥-secreting CD4 ؉ and CD8 ؉ T cells. Reverse transcription-PCR of ears and draining lymph node cells showed the expression of CC chemokines. The dermal model of infection with L. braziliensis herein is able to reproduce aspects of the natural infection, such as the presence of an ulcerated lesion, parasite dissemination to lymphoid areas, and the development of a Th1-type immune response. These results indicate that this model shall be useful to address questions related to the concomitant immunity to reinfection and parasite persistence leading to mucocutaneous leishmaniasis.

Antibody responses to salivary gland sonicate (SGS) from Lutzomyia longipalpis were investigated using serum samples from individuals living in an area where visceral leishmaniasis is endemic. Individuals were classified into 2 groups, according to the alteration of their responses to Leishmania chagasi antigen over the course of 6 months. Group 1 included children who experienced anti-L. chagasi seroconversion from negative to positive; group 2 included children who experienced delayed-type hypersensitivity (DTH) response to L. chagasi antigen conversion from negative to positive. Individuals who experienced seroconversion against L. chagasi antigens did not have increased anti-saliva antibody response, whereas those who developed a positive anti-L. chagasi DTH response had increased immunoglobulin (Ig) G, IgG1 and IgE anti-SGS antibody levels. Despite wide variation, serum samples from individuals in group 2 recognized more bands in SGS than did those from individuals in group 1. This simultaneous appearance of anti-saliva humoral response and anti-L. chagasi cell-mediated immunity supports the hypothesis that induction of immune response against SGS can facilitate induction of a protective response against leishmaniasis.

Visceral leishmaniasis (VL) remains a major public health problem worldwide. This disease is highly associated with chronic inflammation and a lack of the cellular immune responses against Leishmania. It is important to identify major factors driving the successful establishment of the Leishmania infection to develop better tools for the disease control. Heme oxygenase-1 (HO-1) is a key enzyme triggered by cellular stress, and its role in VL has not been investigated. In this study, we evaluated the role of HO-1 in the infection by Leishmania infantum chagasi, the causative agent of VL cases in Brazil. We found that L. chagasi infection or lipophosphoglycan isolated from promastigotes triggered HO-1 production by murine macrophages. Interestingly, cobalt protoporphyrin IX, an HO-1 inductor, increased the parasite burden in both mouse and human-derived macrophages. Upon L. chagasi infection, macrophages from Hmox1 knockout mice presented significantly lower parasite loads when compared with those from wild-type mice. Furthermore, upregulation of HO-1 by cobalt protoporphyrin IX diminished the production of TNF-α and reactive oxygen species by infected murine macrophages and increased Cu/Zn superoxide dismutase expression in human monocytes. Finally, patients with VL presented higher systemic concentrations of HO-1 than healthy individuals, and this increase of HO-1 was reduced after antileishmanial treatment, suggesting that HO-1 is associated with disease susceptibility. Our data argue that HO-1 has a critical role in the L. chagasi infection and is strongly associated with the inflammatory imbalance during VL. Manipulation of HO-1 pathways during VL could serve as an adjunctive therapeutic approach.